PROJECT SUMMARY/ABSTRACT The O-linked glycans displayed on mucins and mucin-like proteins are important for cell-cell communication, homoeostasis, and the maintenance of mucosal barriers. Changes in glycosylation have been linked to cancer and a number of other diseases. In the oral environment, alterations of the O-glycans on salivary mucin MUC7 are associated with oral and systemic pathologies. The ability to detect changes in the O-glycosylation of MUC7 and other mucins is increasingly recognized as important, but the current analytical methods to do so are expensive and technically challenging. Thus, there is a need to develop reagents that can be used like antibodies in relatively simple applications such as blotting, immunohistochemistry or ELISA. An excellent natural source of such reagents are the oral commensal streptococci. Some streptococcal species express adhesins with Siglec-like binding regions (SLBRs) that have high affinity for sialylated glycans. The SLBRs are a unique family of hypervariable glycan-binding proteins that may have evolved to interact with the vast assortment of O-glycan structures displayed on MUC7. We hypothesize that the streptococcal SLBRs are specific for a broader spectrum of human sialoglycan structures, as compared with commercially-available plant lectins, and will therefore greatly enhance the study of MUC7 biology. The overall aim of this project is to determine the extent to which recombinant SLBRs can be used to characterize the differently-glycosylated forms (glycoforms) of MUC7. We will first compare the range of O-glycan sizes and the degree of O- acetylation of terminal sialic acids displayed on MUC7 from a set of five saliva samples that show differential SLBR reactivity and different electrophoretic mobilities. This will demonstrate whether SLBR reactivity is an accurate predictor of MUC7 O-glycan composition, as measured by mass spectrometry. We will then develop an ELISA to determine whether the different MUC7 glycoforms can be easily detected and quantified in human saliva samples, using two SLBRs that have different, well-defined sialoglycan specificities. In addition, to establish a precedent for expanding the set of O-glycan specific reagents, we will identify the ligand for a representative streptococcal SLBR that recognizes a MUC7 O-glycan structure that is apparently more complex than the available synthetic sialoglycans. The combined results will provide the foundation for developing a broader set of O-glycan probes, and for using the SLBRs to purify and analyze MUC7 glycoforms that are otherwise not readily separated by mass or charge. The experiments will also facilitate a better understanding of the breadth of MUC7 glycoform heterogeneity, and the means to assess the physiological and biological effects of O-glycosylation changes. Although the focus is on salivary MUC7, the results will be applicable to the detection, purification and characterization of specific glycoforms of mucins and mucin-like proteins in general.